One of the most unique neural crest populations is the ?cardiac neural crest? that contributes to the outflow tract and outflow septum. Ablation of the cardiac crest in bird embryos causes a heart defect reminiscent of the human birth defect, persistent truncus arteriosus. In preliminary experiments, we have performed a transcriptome analysis of early migrating cardiac neural crest cells, isolated by enhancer-based cell sorting. The results reveal transcription factors (e.g. MafB, Krox20, Lhx1, Id1, Sall3) as well as signaling molecules and other factors that are selectively enriched in the early migrating cardiac neural crest compared to other cell populations. Here, we propose to explore the role of factors identified in our screen in the gene regulatory network that imbues the cardiac neural crest with its unique identify. Loss- and gain-of- function experiments will be used to functionally test the role of these factors and their position in a cardiac crest-specific gene regulatory module. In addition, we will perform cell lineage analysis using retrovirally encoded fluorophores to follow cell fate and gene expression of clonally related cardiac neural crest cells. The following specific aims will be performed: Aim 1: Testing regulatory connections of genes expressed in early migrating cardiac neural crest cells. With our preliminary genome-wide analysis of the active transcriptome of cardiac neural crest cells in hand, we will perform loss-of-function experiments to perturb gene function and establish the order of gene activity in the cardiac neural crest. Starting with MafB, we will perturb function of the transcription factors and analyze effects on expression of known neural crest genes as well as new genes uncovered in our screen. In this way, we can assemble a functional gene battery in the early migratory cardiac neural crest. Aim 2: Transcriptional profiling of individual cardiac neural crest cells using single cell RNA-seq and multiplex single molecule fluorescent in situ hybridization (smFISH). To gain a comprehensive view of the gene expression profile of individual cardiac crest cells, we will perform single cell RNA-seq on several hundred cells per time point sorted from the cardiac crest. To perform a similar analysis with the advantage of providing spatial information, we have devised an adaptation of smFISH called Spatial Genomic Analysis (SGA) that will be performed on tissue sections of carefully staged embryos, enabling simultaneous analysis of the expression of 35 probes selected from cardiac crest genes identified in our transcriptome dataset. Aim 3: Retrovirally mediated clonal analysis coupled with Spatial Genomic Analysis (SGA) to examine the cell lineage and fate of individual chick cardiac neural crest. To determine the developmental potential of individual cardiac neural crest cells to contribute to the cardiovascular system, we will perform multi-color clonal analysis of the cardiac neural crest region of chick embryos using a mixture of recombinant replication incompetent avian retroviruses (RIA) encoding different fluorescent proteins to label individual clones with distinct colors. By coupling clonal analysis with SGA, we will determine at single cell resolution which cells co-express transcription factors and signaling molecules identified in our screen. These specific aims are designed to define the molecular and cellular mechanisms underlying cardiac neural crest development. The ultimate goal is to provide important insights into the pathogenesis of septal defects that will lead to development of novel strategies for the prevention of neural crest-related heart defects.